The present invention relates to an array of hybridisation probes, use of hybridisation probes, a method of determining hybridisation of an array of such probes and methods for characterising cDNA and sequencing nucleic acid.
Mass spectrometry is a highly sensitive technique for determining molecular masses, so sensitive that it can be used to give detailed structural information as well. Essentially, the molecule(s) to be analysed is vaporised and ionised into a vacuum. The vapor phase ions are accelerated through electromagnetic fields and their mass/charge ratio is determined by analysis of the molecules behaviour in the electromagnetic fields. Various mass spectrometry technologies exist determined by the main targets of the systems or on the various ionisation techniques that they employ. On the whole mass spectrometry is used for direct analysis of molecules in order to determine their mass; identify them or acquire structural information. (For a textbook on mass spectrometry see reference 1)
Combinatorial chemistry (for a review of this field see reference 2) has lead to more specific requires for indirect analysis of molecules. Various strategies now exist to generate large numbers of related molecules, using solid phase synthesis techniques, in a combinatorial manner. Since most systems generate individual molecules on beads, these can be screened for desirable properties. However, it is often the case that molecule being screened is not directly recoverable or difficult to analyse directly for other reasons so indirect labelling of beads and hence their molecules has been proposed as a solution. Most techniques for xe2x80x98encodingxe2x80x99 (see reference 3) combinatorial libraries seem to involve using labels that are in some sense capable of being xe2x80x98sequencedxe2x80x99 (see reference 4), for example amino acids and nucleic acids are often used to encode libraries because the technologies to sequence these are routine and relatively rapid for short peptides and oligonucleotides, an analysis that is often also performed by mass spectrometry these days. Other organic entities are sequencable such as halogenated benzenes and secondary amides and can be used for these purposes (see references 5 and 6).
An alternative approach (see reference 7) uses a variety of combinatorial monomers that can be enriched in particular isotopes to generate labels that give unique isotope signatures in a mass spectrum. This approach allows the generation of large numbers of labels that have distinct patterns of isotope peaks in restricted regions of the mass spectrum. This method is ideal for uniquely identifying a single compound whose bead has been isolated from a large combinatorial library, for example but would almost certainly have problems resolving large numbers of molecules simultaneously.
References 15 to 17 disclose applications of mass spectrometry to detect binding of various ligands.
The present invention provides an array of hybridisation probes, each of which comprises a mass label linked to a known base sequence of predetermined length, wherein each mass label of the array, optionally together with the known base sequence, is relatable to that base sequence by mass spectrometry. Preferably, each of the hybridisation probes comprises a mass label cleavably linked to a known base sequence of predetermined length, wherein each mass label of the array, when released from its respective base sequence, is relatable to that base sequence by mass spectrometry, typically by its mass/charge ratio which is preferably uniquely identifiable in relation to every other mass label in the array.
The present invention further provides use of a hybridisation probe, comprising a mass label linked to a known base sequence of predetermined length, in a method for determining hybridisation of the probe by mass spectrometry of the mass label optionally together with the known base sequence. Preferably, the hybridisation probe comprises a mass label cleavably linked to a known base sequence of predetermined length.
The present invention further provides a method for determining hybridisation of a probe with a target nucleic acid, which method comprises
(a) contacting target nucleic acid with a hybridisation probe, which comprises a mass label linked to a known base sequence of predetermined length, under conditions to hybridise the probe to the target nucleic acid and optionally removing unhybridised material; and
(b) identifying the probe by mass spectrometry.
The present invention further provides a method for determining hybridisation of an array of probes with a target nucleic acid, which method comprises
(a) contacting target nucleic acid with each hybridisation probe of the array under conditions to hybridise the probe to the target nucleic acid, and optionally removing unhybridised material, wherein each probe comprises a mass label linked to a known base sequence of predetermined length; and
(b) identifying the probe by mass spectrometry.
Preferably, the or each mass label is cleavably linked to its respective known base sequence and each hybridised probe is cleaved to release the mass label, which released label is identified by mass spectrometry.
The predetermined length of the base sequence is usually from 2 to 25.
Each mass label may be cleavably linked to the known base sequence by a link which may be a photocleavable link, a chemically cleavable link or a thermally cleavable link. According to one embodiment, the link cleaves when in a mass spectrometer, for example in the ionisation chamber of the mass spectrometer. This has the advantage that no cleavage of the link need take place outside of the mass spectrometer. By appropriate selection of the link, cleavage is effected in the mass spectrometer so as to afford a rapid separation of the known base sequence from the mass label so that the mass label can be readily identified. The link is preferably less stable to electron ionisation than the mass label. This allows cleavage of the link without fragmentation of any part of the mass label inside the mass spectrometer.
In a preferred embodiment, the mass label is stable to electron ionisation at 50 volts, preferably at 100 volts. Conditions of electron ionisation occurring in mass spectrometers can cause fragmentation of molecules and so it is convenient to measure stability of a mass label in terms of its ability to withstand electron ionisation at a particular voltage. Stability to electron ionisation is also a useful guide as to stability of the molecule under collision induced dissociation conditions experienced in a mass spectrometer.
Preferably, the mass labels are resolvable in mass spectrometry from the known base sequences. This is advantageous because the need to separate or purify each mass label from their respective base sequences is avoided. Accordingly, in a preferred embodiment, the mass label and the known base sequences are not separated before entry into the mass spectrometer.
In a further preferred embodiment, the method is exclusively in line. By in-line is meant that at no stage in the method is there a step which is performed off-line. This is advantageous because the method can be performed as a continuous method and may be readily automatable.
In one embodiment, each mass label is designed to be negatively charged under ionisation conditions. This has the advantage that buffer conditions can be arranged whereby nucleic acid accompanying the mass label is positively charged. When in a mass spectrometer, this enables ready separation of the mass label from the DNA and results in less background noise in the mass spectrum.
Preferably the known base sequence has linked thereto a plurality of identical mass labels. Using a plurality of identical mass labels has the advantage that simultaneous cleavage or the plurality of mass labels gives rise to a higher signal because a higher concentration of mass labels may be measured.
In one embodiment, the known base sequence comprises a sticky end of an adaptor oligonucleotide containing a recognition site for a restriction endonuclease which cuts at a predetermined displacement from the recognition site.
This invention advocates the use of labels with well-behaved mass spectrometry properties, to allow relatively large numbers of molecules to be identified in a single mass spectrum. Well behaved meaning that the molecules minimise the number of peaks that they generate in a spectrum by preventing multiple ionisation states and not using especially labile groups. Several decades of mass spectrometry in organic chemistry has identified certain molecular features that are favorable for such use and certain features to be avoided.
It is possible to label molecules particularly biological molecules with mass, as an indicator of the molecules identity. A code relating a molecule""s mass to its identity is easy to generate, e.g. given a set of molecules which it is desirable to identify one can simply select an increasing mass for each distinct molecule to be identified. Obviously many molecules can be identified on the basis of their mass alone and labelling may seem superfluous. It may be the case that certain sets of molecules, although unique, may have closely related masses and be multiply ionisable, making resolution in the mass spectrometer difficult hence the utility of mass-labelling. This is particularly true of nucleic acids which are often isobaric but still distinct, e.g. the sequence TATA is distinct from TTAA, TAAT, etc. but in a mass spectrometer these would be difficult to resolve. Furthermore one might like the molecules to be identified to perform a certain function as well as being detectable and this means direct detection might be impossible so a removable label that can be independently detected is of great utility. This will allow large numbers of molecules that may be very similar to be analysed simultaneously for large scale screening purposes.
This invention describes the use of libraries of mass labels which identify the sequence of a covalently linked nucleic acid probe. The construction of mass labels is relatively simple for a qualified organic chemist. This makes it easy to produce labels that are controllably removable from their respective probe and which have beneficial physical properties that aid ionisation into a mass spectrometer and that aid detection and resolution of multiple labels over a large range of relative quantities of those labels.